Vaccinia virus has a genome of 185,000 bp that encodes approximately 200 polypeptides. These genes are expressed in a coordinated fashion so that some polypeptides are made before and others after DNA replication. One vaccinia virus gene that is expressed throughout the growth cycle was found to have two RNA start sites about 55 bp apart. The site nearest to the coding segment was used at early times in infection and the other was used at late times. In vitro mutagenesis studies revealed that there are two independent promoters and that the regulatory signals are located within 31 bp of each RNA start site. RNA polymerase subunits, synthesized in reticulocyte lysates programmed with early vaccinia virus mRNA, were immunoprecipitated by antibody prepared against the purified enzyme. The subunit genes were mapped by hybridization selection of mRNA to cloned DNA fragments prior to translation. The genes for 2 subunits of 147 kD and 22 kD were sequenced. The large subunit was shown to have considerable homology with the Beta' subunit of E. coli RNA polymerase. A soluble extract capable of selectively transcribing added early vaccinia virus genes was prepared by disrupting purified vaccinia virus particles. Correct initiation, termination and polyadenylation were demonstrated and the signals for each were defined by transcription of truncated templates. Termination was shown to occur about 50 bp downstream of the signal.